Protein modification by ubiquitin plays important roles in such processes as cell cycle control, DNA repair, ribosome biogenesis, the heat shock response, and intracellular protein degradation. Substrates include cytoplasmic, nuclear, and integral membrane proteins. The diverse functions of ubiquitin generally involve the activity of distinct ubiquitin-conjugating enzymes. We have found that the conjugating enzymes E2-20K and E2-230K appear to be expressed only in the erythroid lineage. The discovery of ubiquitin-conjugating enzymes expressed in a tissue-specific fashion suggests that the functions of ubiquitination involve not only general cellular processes (above), but extend to specific differentiative events as well. The differentiation of erythroid cells is a global remodelling process whose mechanism is poorly understood. Because the levels E2-20K and E2-230K are high in the reticulocyte rather than the erythrocyte, it is likely that these enzymes represent a novel class of erythroid proteins which do not function in the mature erythrocyte but rather in the differentiative process that gives rise to it. It is proposed that E2-20K and E2-230K provide functions critical for erythroid differentiation. This hypothesis will be tested as follows. The genes for E2-20k and E2-230K will be cloned, sequenced, and used to generate murine erythroleukemia (MEL) cell derivatives in which these genes have been deleted by homologous recombination. We will then test whether such mutations affect the process of erythroid differentiation as represented in this model system. Potential conjugative and degradative targets of these E2's will be identified as specific proteins whose ubiquitination or turnover drifters between mutant and parental cells. A long-term goal is to relate the observed differentiative phenotypes to the nature of the observed substrates. The results are expected to be highly novel in providing the first insights into roles of ubiquitin in development. In addition, the experiments are expected to yield major insights into the mechanism of erythroid differentiation, with possible applications in the understanding of congenital diserythropoietic anemias.